Limitations of Technology in Quantitative Microscope
Where photo- or video-microscopy are employed as analytical techniques, a light standard providing preset levels of illumination over a homogeneous field covering the viewing area of a microscope is a useful accessory for system validation and testing. Functional studies of cell biology routinely seek information about cellular activity by means of video imaging, using image sequences to monitor correlated cellular actions, such as calcium flux transients in relation to force generation in muscle cells. Quantitative information concerning intracellular ion concentration changes is obtained using ratio imaging techniques, permitting degrees of cellular activity to be compared. The reliability of photomicroscopic assays depends on accurate translation of light intensities, some of which are exceedingly faint, into pixel brightness levels. Unfortunately, video detection systems and accessories employed for low light recording cause distortions of both brightness and geometry in the image. Photoelectron multipliers in SIT and intensified SIT cameras exhibit non-linear response at light levels below saturation. In addition to non-linearity, the response of electrostatically focused SIT cameras is greater in the center of the recorded field. This defect, known as “shading”, can vary in magnitude with average illumination in some cameras. The microscope light collection and illumination paths can also contribute to shading in the image. These effects complicate the task of insuring the photometric accuracy of images obtained under different recording conditions. Newer solid-state microchannel plate intensifiers used with CCD cameras, which are free from shading effects, still are non-linear and can impose onto the low light image a repeating pattern of varying intensity (chicken wire pattern) caused by the array of microchannels. This problem has been largely overcome in the most recent intensifiers. The newest of methods employing light integration with CCD cameras are by far the most linear and free from spatial distortion than prior art designs. Integration provides the ability to image well in low light, however, the temporal resolution is compromised. Very high speed imaging systems employing two-dimensional photodiode arrays without CCD architecture, such as Neuroplex™ (Red Shirt Imaging LLC, Fairchild, Conn.), have been successfully used to monitor fast dynamics. These imagers represent a compromise between high spatial resolution and speed while providing good linearity and sensitivity. The several forms of image distortion associated with the microscope and the light detection systems must be identified and corrected, or at least shown to be insignificant, before quantitative information can be obtained using photomicroscopy.
Applications for Light Standards in Quantitative Microscopy
Aside from assessments of the performance of microscope apparatus, the main applications for a light standard in microscopy are in facilitating interpretation of quantitative data. In the life sciences, fluorescent probes of cell function produce light signals that are linked directly with ion concentrations and other cellular activity. Examples of additional areas of use are:
Cell calcium recordings—Fluorescent calcium indicators are used for quantitation of intracellular calcium concentration and time dependent changes in concentration. Fluo-3, Calcium Green, Fura Red etc. respond to calcium by changing the fluorescent yield within a single emission band without significant wavelength shifts.
Concentration must be determined by careful comparison of indicator responses under conditions of zero and high intracellular calcium, usually obtained after experimental maneuvers, or by comparison of cell readings with fluorescence from known calcium references. These calibrations do not in themselves require a standard light reference. The light standard aids this measurement by checking excitation levels to insure illumination level is safe, i.e. below a level known to give phototoxic effects, and to form a base standard that ensures repeatable experiments using the same standard. Once reproducibility is obtained, it becomes possible, under conditions where dye entering the cell attains its fluorescent property, to monitor the degree of dye loading.
Thus, the light standard facilitates reproducible experimental conditions. Calcium responses can also be compared with the light standard output, and in turn, with each other. In addition to single wavelength indicators, the calcium dye Fura-2 responds by alternating between two UV excitation bands as calcium is bound and released from the indicator. Indo-1 responds by shifting its peak fluorescence emission from 495 nm to 405 nm upon binding calcium. The calcium response is determined by the ratio of fluorescence intensities at these wavelengths. Although the signals from Fura-2 would be assessed similarly to those of the single wavelength dyes, signals from Indo-1 and Calcium Green/Fura Red dye combinations used for ratiometric recording would be assessed using light standards at two different wavelengths. For a standard based on light emitting diode (LED) sources, the calcium response indicated by the fluorescence ratio would be compared to ratios generated from the light output of blue and green LEDs (Indo-1) or green and red LEDs (Calcium Green/Fura Red). Dye fluorescence signals can be simulated if both wavelengths are combined and emitted simultaneously.
Potentiometric recordings—Voltage-sensitive dyes are also available with single wavelength and dual-wavelength responses. The most used dual-wavelength voltage dye, di-8-ANEPPS, responds with graded shifts in both excitation and emission spectra which are proportional to change in the transmembrane and membrane dipole potentials. Spectral shifts represent a departure from pure intensity changes because the fluorescence ratio is independent of the absolute fluorescence from the cell (assuming that all the dye is in the membrane being evaluated). Therefore reference light ratios from a standard can be used to compare voltage responses between different experiments and apparatus.
Prior Art of Standard Light Sources
To aid in assessing the optical performance of photo- and video-microscopes as well as the reproducibility of recordings, chemical standard light sources have been developed which are based on fluorescent microbeads, solid blocks and fluorescence filled glass capillary tubes. Beads permit the user to determine the degree to which the optical system faithfully images small three dimensional objects. Solid standards, such as uranyl glass and polymethacrylate blocks containing anthracene or rhodamine B (Starma Cells Inc.), undergo almost no photobleaching and are thus excellent tools to monitor fluctuations in excitation light levels. Solid standards have also been used to generate uniformly fluorescent fields from which to correct camera shading functions. Capillary tubes or cuvettes filled with known concentrations of fluorophores are used to characterize camera linearity, and can provide light intensities in absolute units with known excitation power, although in some cases the emission must be corrected for anisotropy. Thus, primary uses for light standards have traditionally been 1) provision of a light intensity “benchmark” for comparison between different setups, 2) assessment of the point spread or modulation transfer function, 3) assessment of detector linearity and sensitivity to a known intensity, 4) monitoring of the microscope's own illumination.
As useful as they are for quantitative work, chemical standards present some practical difficulties. Solutions must be duplicated exactly to serve as calibration light sources; thus their preparation is both time consuming and difficult and most solution standards show moderate photobleaching with use. Furthermore, in all of these standards light output is subject to change as the excitation source power changes. Thus, lamp noise from power fluctuations and aging of the burner introduce errors. Lastly, chemical standards cannot be implemented with features that allow a programmed presentation of light intensities for specific modes of operation that are useful for instrument setup, calibration, and simulation of actual measurements.
Prior Art in the Electronic Light Standard
In order to circumvent some of the problems of chemical light standards, a device employing light bars and optical feedback control of light output was designed and is disclosed in U.S. Pat. No. 5,489,771. The light bar is a style of light emitting diode (LED) that emits through a plastic diffuser, producing a relatively uniform light output. Radiance from a 1 mm circular output aperture on the diffuser surface directly in line with the internal LED chip was constant to within 2.5%. The light bar produced 6–18 mcd maximum optical power at 20 mA forward current. To avoid temperature dependant changes in emission spectra, the earlier electronics did not drive the light bar above 50% of maximum rated power dissipation. The diffuser caused over 90% of the light produced from the internal LED chip to be scattered outside the central region of uniform radiance. Some of this light was intercepted by a photodiode that provided the signal for feedback control of the light intensity. Less than 10% of the optical power passed through the output aperture, resulting in less than 2 mcd maximum output from the light standard.
The early system combined red, yellow and green light bars, each with their own output aperture, onto a holder that could be placed on the microscope stage. Output of each light bar could be set independently from a controller to produce five equally spaced intensities. Although the original device provided good performance in terms of intensity, wavelength coverage and light output uniformity, it still had several drawbacks. Light power output was insufficient for comparison with intensities produced in bright-field images and in fluorescence from many brighter dye probes such as di-8-ANEPPS and GFP. Additionally, spatial uniformity suffered from the fact that the light bar directly illuminated the aperture through a diffuser, producing a symmetric non-uniformity around the center. Therefore, not only were light bars of relatively low light conversion efficiency and power output, the light of different wavelengths could not be combined into a common output aperture. There was no provision for assessing the output of the microscope illuminator. Lastly, manual rather than programmable output settings limited the versatility of the prior art light standards.
Camera test light sources are commercially available from Advanced Illumination Inc. and can be purchased with mutli-color output capability. One light source provides a backlit field of illumination from an array of LEDs over areas of several square inches. Unevenness of the light is specified at approximately 8% over the full field. A second light source by the same company employs a hemispherical reflecting surface to produce an illuminated field with approximately the same degree of evenness and field coverage as that of the LED array illuminator. Neither system employs optical feedback control of light intensity; instead using a precision regulated current supply. Since the systems are not intended as light standards for microscopy, they are not physically adapted to mount on a microscope. Although the second system of Advanced Illumination employs a reflecting surface in the shape of a hemisphere, this design does not comprise an integrating chamber that is capable of high spatial output uniformity. Finally, neither system provides programmable test features.